Objectives: The aim of the study is to describe explain in detail the molecular method for the diagnosis of the Mycoplasma genitalium in the Institute of Public Health of the Split-Dalmatia County and sampling methods for diagnosing infections caused by the M. genitalium bacteria. Material and methods: The procedure of molecular diagnosis of M. genitalium is based on the detection and amplification of nucleic acid using the real-time PCR method. The Cobas z480 analyzer and the LightMix Kit Mycoplasma Genitalium are used for the diagnostic purpose of determining M. genitalium in the Teaching Institute of Public Health of Split-Dalmatia County. Kits can detect 10 equivalent copies of the bacterium's genome. Samples are cervical and urethral swabs and the first-void urine. Before amplification, samples are placed in an extraction device to obtain purified nucleic acid for the PCR method. Results: A positive test result indicates the presence of bacterial DNA in the tested sample, while a negative result indicates that bacterial DNA was not detected in the sample. Conclusion: Mycoplasma genitalium is a sexually transmitted bacterium that most often causes asymptomatic infections. Symptomatic infections include increased discharge from the genitals, dysuria, lower abdominal pain, intermenstrual bleeding, etc. A growing problem is the development of bacterial resistance to antimicrobial drugs. In case of infection, it is necessary to treat partners of infected persons at the same time. Untreated infections can lead to the development of complications (infertility, pelvic inflammatory disease, ectopic pregnancy, premature birth...).